Abstract
RNA-Seq using second-generation sequencing technologies permits generation of a reference unigene set for a given species, in the absence of a well-annotated genome sequence, supporting functional genomics studies, gene characterisation and detailed expression analysis for specific morphophysiological or environmental stress response traits. A reference unigene set for lentil has been developed, consisting of 58,986 contigs and scaffolds with an N50 length of 1719 bp. Comparison to gene complements from related species, reference protein databases, previously published lentil transcriptomes and a draft genome sequence validated the current dataset in terms of degree of completeness and utility. A large proportion (98%) of unigenes were expressed in more than one tissue, at varying levels. Candidate genes associated with mechanisms of tolerance to both boron toxicity and time of flowering were identified, which can eventually be used for the development of gene-based markers. This study has provided a comprehensive, assembled and annotated reference gene set for lentil that can be used for multiple applications, permitting identification of genes for pathway-specific expression analysis, genetic modification approaches, development of resources for genotypic analysis, and assistance in the annotation of a future lentil genome sequence.
Highlights
The application of next-generation sequencing (NGS) technologies to sequence mRNA (RNA-Seq) can provide comprehensive characterisation of the gene-space of a given organism, allowing definition of an extensive gene catalogue, identification of specific coding DNA sequences (CDSs), development of gene-associated genetic markers, comparative genomics analysis and quantification of gene expression [1]
96% of the transcripts were found to be common in reciprocal BLAST searches, indicating that the previously published lentil assemblies are fragmented in nature
The improved characteristics of the most recent lentil and field pea transcriptome assemblies reflect the superior performance of more contemporary sequencing platforms, with consequent higher value for functional genomics and molecular breeding applications
Summary
The application of next-generation sequencing (NGS) technologies to sequence mRNA (RNA-Seq) can provide comprehensive characterisation of the gene-space of a given organism, allowing definition of an extensive gene catalogue, identification of specific coding DNA sequences (CDSs), development of gene-associated genetic markers, comparative genomics analysis and quantification of gene expression [1]. Regular advances in NGS technologies have removed the impediments to characterisation of the transcriptomes and genomes of crop species with relatively lower scales of cultivation and economic value, such as lentil (Lens culinaris Medik.) [2,3,4]. Lentils provide a rich and inexpensive source of protein, carbohydrates, micronutrients and vitamins and are a major source of nutrition in developing nations. Lentils play a beneficial role in agriculture through nitrogen fixation and can assist in management of weeds and pathogen through crop rotation
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